EP0214057A1 - Lectin attachment factors of Neisseria gonorrhoeae, method for their preparation and therapeutic composition containing these lectins, the corresponding antibodies or the corresponding attachment receptors - Google Patents
Lectin attachment factors of Neisseria gonorrhoeae, method for their preparation and therapeutic composition containing these lectins, the corresponding antibodies or the corresponding attachment receptors Download PDFInfo
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- EP0214057A1 EP0214057A1 EP86401916A EP86401916A EP0214057A1 EP 0214057 A1 EP0214057 A1 EP 0214057A1 EP 86401916 A EP86401916 A EP 86401916A EP 86401916 A EP86401916 A EP 86401916A EP 0214057 A1 EP0214057 A1 EP 0214057A1
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 241000588652 Neisseria gonorrhoeae Species 0.000 title claims abstract description 16
- 102000004856 Lectins Human genes 0.000 title abstract description 6
- 108090001090 Lectins Proteins 0.000 title abstract description 6
- 239000002523 lectin Substances 0.000 title abstract description 6
- 239000000203 mixture Substances 0.000 title description 3
- 238000002360 preparation method Methods 0.000 title description 3
- 230000001225 therapeutic effect Effects 0.000 title description 2
- 235000000346 sugar Nutrition 0.000 claims abstract description 28
- 239000000284 extract Substances 0.000 claims abstract description 11
- 230000001936 parietal effect Effects 0.000 claims abstract description 7
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 11
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 10
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 10
- 229960002442 glucosamine Drugs 0.000 claims description 10
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 9
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 8
- 229920000936 Agarose Polymers 0.000 claims description 7
- 241000588653 Neisseria Species 0.000 claims description 6
- 238000000760 immunoelectrophoresis Methods 0.000 claims description 6
- 230000000890 antigenic effect Effects 0.000 claims description 5
- 235000009508 confectionery Nutrition 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 4
- 230000003100 immobilizing effect Effects 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 102100034612 Annexin A4 Human genes 0.000 claims 1
- 108090000669 Annexin A4 Proteins 0.000 claims 1
- 239000004480 active ingredient Substances 0.000 claims 1
- 238000003018 immunoassay Methods 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 7
- 102000036639 antigens Human genes 0.000 abstract description 7
- 108091007433 antigens Proteins 0.000 abstract description 7
- 150000008163 sugars Chemical class 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000003449 preventive effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 2
- 206010018612 Gonorrhoea Diseases 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000001989 nasopharynx Anatomy 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- WCWOEQFAYSXBRK-SVZMEOIVSA-N (3r,4s,5r,6r)-2-amino-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound NC1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O WCWOEQFAYSXBRK-SVZMEOIVSA-N 0.000 description 1
- UTVXFQMLZSPQLB-DHVFOXMCSA-N (3s,4r,5s,6s)-2-amino-6-methyloxane-3,4,5-triol Chemical compound C[C@@H]1OC(N)[C@@H](O)[C@H](O)[C@@H]1O UTVXFQMLZSPQLB-DHVFOXMCSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 101000794863 Neisseria gonorrhoeae Anthranilate synthase component 1 Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000009109 curative therapy Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 230000000503 lectinlike effect Effects 0.000 description 1
- 125000003071 maltose group Chemical group 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000003708 urethra Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/561—Immunoelectrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to lectin-attachment factors of Neisseria gonorrhoeae as well as the application of certain immobilized receptors for the preparation of these factors and their demonstration or their determination.
- Neisseria gonorrhoeae of various origins strains from different reference collections as well as strains responsible for localized gonococcal diseases and strains responsible for disseminated gonococcal diseases .
- Neisseria gonorrhoeae of the lectin type have been able to be demonstrated thanks to their property of adhesion to immobilized sweet receptors, in particular receptors of the glucosamine or maltose type.
- lectins-attachment factors due to their antigenic property, and / or their lectin-like activity, can be used in these pharmaceutical compositions, themselves or the corresponding antibodies, either for curative or preventive purposes. , for the treatment or especially the prevention of gonococcal infections and also of infections mixed with other bacteria, anaerobic for example.
- Immobilized sweet receptors which can be used in the context of this process, mention should be made more particularly of the sweet receptors immobilized on an agarose support of the glucosamine, maltose type. Immobilized receptors can be in any form; in particular, they can be used in the form of a column or else implemented by "batch".
- binding properties of these lectin-attachment factors and their specificity make it possible to envisage methods of assaying or demonstrating Neisseria gonorrhoeae, in particular in pathological products.
- Neisseria gonorrhoeae will include at least one step of fixing the lectin-attachment factors of Neisseria g onorrhoeae on a receptor sugar of glucosamine or maltose types, this receptor sugar preferably being immobilized on a support such as an agarose support.
- attachment factor lectins Since the attachment factor lectins have antigenic properties, it is possible to demonstrate them and / or to measure them by immunological methods, in particular by immunoelectrophoresis and immunoenzymatic methods.
- immunological methods implementing the antigen / antibody reactions can be of very varied types, it can in particular be immunoelectrophoresis such than line-absorption type immunoelectrophoresis or any other immunological method well known to those skilled in the art.
- the purpose of this example is to demonstrate the lectins-attachment factors described above.
- the following seven sugars have been tested: maltose, galactosylamine, fucosylamine, glucosamine, acetylglucosamine, D galactosamine, thiogalactoside.
- These sugars were fixed on agarose beads by techniques known to those skilled in the art.
- a bacterial suspension of C, 5 ml was brought into contact with C, 5 ml of sugar immobilized on beads. After incubation, the beads were washed then stained and observed under a microscope. According to the sugars, two cases arose.
- Neisseria gonorrhoeae adhered to these beads.
- no adhesion of the gonococcus was observed.
- the possibility of inhibiting the fixing of the gonococcus was studied by incubating beforehand the suspension of Neisseria gonorrhoeae with a free sugar corresponding to the fixed sugar used in the following step. The results observed with Reference strains from two collections and wild strains are identical and demonstrate the specific binding of these lectin-attachment factors.
- Fixation was in fact inhibited only if the free sugar corresponding to the incubation mixture was added in sufficient concentration (25 mg / ml). Inhibition was not achieved with any other sugar. Therefore, there are lectins-attachment factors on the surface of the gonococcus which allow adhesion to immobilized receptors. These have among others glucosamine and maltose residues. This allows the purification of these factor lectins by affinity chromatography on immobilized sugar receptors.
- the wells contained the following immobilized sugars: well 3: glucosamine, well 7: galactosamine, well 8: maltose.
- well 3 glucosamine
- well 7 galactosamine
- well 8 maltose.
- the results obtained are identical: absorption of the precipitation lines with immobilized sugars maltose glucosarin only.
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- Proteomics, Peptides & Aminoacids (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
La présente invention concerne des lectines-facteurs d'attachement de Neisseria gonorrhoeae ainsi que l'application de certains récepteurs immobilisés pour la préparation de ces facteurs et leur mise en évidence ou leur dosage.The present invention relates to lectin-attachment factors of Neisseria gonorrhoeae as well as the application of certain immobilized receptors for the preparation of these factors and their demonstration or their determination.
Plus particulièrement, il s'agit de la mise en évidence et de la caractérisation de lectines-facteurs d'attachement chez Neisseria gonorrhoeae d'origines diverses : souches de différentes collections de référence ainsi que souches responsables de gonococcies localisées et souches responsables de gonococcies disséminées.More particularly, it involves the identification and characterization of lectin-attachment factors in Neisseria gonorrhoeae of various origins: strains from different reference collections as well as strains responsible for localized gonococcal diseases and strains responsible for disseminated gonococcal diseases .
Ces facteurs d'attachement de Neisseria gonorrhoeae du type lectine ont pu être mis en évidence grâce à leur propriété d'adhésion à des récepteurs immobilisés sucrés, en particulier des récepteurs de type glucosamine ou maltose.These attachment factors of Neisseria gonorrhoeae of the lectin type have been able to be demonstrated thanks to their property of adhesion to immobilized sweet receptors, in particular receptors of the glucosamine or maltose type.
La fixation des sucres précédents sur des supports immobilisants tels que l'agarose permet de purifier les lectines-facteurs d'attachement. Ces lectines-facteurs d'attachement, en raison de leur propriété antigénique, et/ou de leur activité de type lectine, peuvent être utilisées dans ces compositions pharmaceutiques, elles-mêmes ou les anticorps correspondants, soit à titre curatif, soit à titre préventif, pour le traitement ou surtout la prévention des infections gonococciques et également des infections mixtes avec d'autres bactéries, anaérobies par exemple.The fixing of the preceding sugars on immobilizing supports such as agarose makes it possible to purify the lectins-attachment factors. These lectin-attachment factors, due to their antigenic property, and / or their lectin-like activity, can be used in these pharmaceutical compositions, themselves or the corresponding antibodies, either for curative or preventive purposes. , for the treatment or especially the prevention of gonococcal infections and also of infections mixed with other bacteria, anaerobic for example.
Ces lectines-facteurs d'attachement sont particulièrement intéressantes étant donné la recrudescence mondiale actuelle de la gonococcie et de ses forme asymptomatiques. Ces lectines-facteurs d'attachement pourront entraîner une rupture de la chaîne épidémiologique soit par l'établissement d'un diagnostic de laboratoire, soit, et surtout, par la mise au point d'un produit pharmaceutique préventif.These lectin-attachment factors are particularly interesting given the current worldwide resurgence of gonococcal disease and its asymptomatic forms. These lectins-attachment factors could cause a break in the epidemiological chain either by establishing a laboratory diagnosis, or, above all, by developing a preventive pharmaceutical product.
La présente invention concerne également un procédé de préparation de ces lectines-facteurs d'attachement de Neisseria gonorrhoeae, caractérisé en ce que :
- a) des extraits protéiques de la membrane externe de Neisseria gonorrhoeae sont préparés puis solubilisés,
- b) ces extraits sont mis en présence de récepteurs sucrés immobilisés,
- c) puis les lectines-facteurs d'attachement ainsi fixées aux récepteurs sont séparés.
- a) protein extracts from the external membrane of Neisseria gonorrhoeae are prepared and then dissolved,
- b) these extracts are placed in the presence of immobilized sugar receptors,
- c) then the lectins-attachment factors thus attached to the receptors are separated.
Parmi les récepteurs sucrés immobilisés utilisables dans le cadre de ce procédé, il faut citer plus particulièrement les récepteurs sucrés immobilisés sur support agarose de type glucosamine, maltose. Les récepteurs immobilisés peuvent se présenter sous une forme quelconque; en particulier, ils peuvent être utilisés sous forme de colonne ou bien mis en oeuvre par "batch".Among the immobilized sweet receptors which can be used in the context of this process, mention should be made more particularly of the sweet receptors immobilized on an agarose support of the glucosamine, maltose type. Immobilized receptors can be in any form; in particular, they can be used in the form of a column or else implemented by "batch".
Enfin, les propriétés de fixation de ces lectines-facteurs d'attachement et leur spécificité permettent d'envisager des procédés de dosage ou de mise en évidence de Neisseria gonorrhoeae notamment dans les produits pathologiques.Finally, the binding properties of these lectin-attachment factors and their specificity make it possible to envisage methods of assaying or demonstrating Neisseria gonorrhoeae, in particular in pathological products.
Ces procédés de mise en évidence de Neisseria gonorrhoeae comporteront au moins une étape de fixation des lectines-facteurs d'attachement de Neisseria gonorrhoeae sur un sucre récepteur de types glucosamine ou maltose, ce sucre récepteur étant de préférence immobilisé sur un support tel qu'un support d'agarose.These methods of demonstrating Neisseria gonorrhoeae will include at least one step of fixing the lectin-attachment factors of Neisseria g onorrhoeae on a receptor sugar of glucosamine or maltose types, this receptor sugar preferably being immobilized on a support such as an agarose support.
Les lectines-facteurs d'attachement présentant des propriétés antigéniques, il est possible de les mettre en évidence et/ou de les doser par des procédés immunologiques, en particulier par des procédés d'immunoélectrophorèse et immunoenzymatique.Since the attachment factor lectins have antigenic properties, it is possible to demonstrate them and / or to measure them by immunological methods, in particular by immunoelectrophoresis and immunoenzymatic methods.
Dans le cadre d'un dosage de type immunologique, on pourra doser ou mettre en évidence la présence de Neisseria gonorrhoeae dans un prélèvement en recherchant la disparition du précipité caractéristique antigène/ anticorps dans le prélèvement soumis préalablement à un traitement afin de libérer ces lectines-facteurs d'attachement de la membrane externe du gonocoque et à l'action d'un sucre récepteur fixant lesdites lectines-facteurs d'attachement.Within the framework of an immunological type assay, it will be possible to assay or demonstrate the presence of Neisseria gonorrhoeae in a sample by seeking the disappearance of the characteristic antigen / antibody precipitate in the sample subjected to a treatment in order to release these lectins- attachment factors of the gonococcal outer membrane and the action of a receptor sugar fixing said lectins-attachment factors.
Il est également possible de metre en évidence les précipités spécifiques antigènes/anticorps correspondant aux lectines-facteurs d'attachement à partir du prélèvement ayant été soumis au traitement d'extraction de ces facteurs pariétaux puis ayant été traités par des récepteurs sucrés immobilisés, lesdits récepteurs sucrés immobilisés étant séparés puis remis en solution pour libérer les éventuelles lectines-facteurs d'attachement.It is also possible to highlight the specific antigen / antibody precipitates corresponding to the lectins-attachment factors from the sample having been subjected to the extraction treatment of these parietal factors and then having been treated with immobilized sugar receptors, said receptors immobilized sugars being separated and then put back into solution to release any lectins-attachment factors.
Bien entendu, la présence ou au contraire l'absence de ces lectines-facteurs d'attachement caractérisées par des méthodes immunclogiques mettant en oeuvre les réactions antigènes/anticorps peuvent être de types très variés, il peut en particulier s'agir d'immunoélectrophorèse telle que l'immunoélectrophorèse de type "line-absorption" ou toute autre méthode immunologique bien connue de l'homme de métier.Of course, the presence or on the contrary the absence of these lectin-attachment factors characterized by immunological methods implementing the antigen / antibody reactions can be of very varied types, it can in particular be immunoelectrophoresis such than line-absorption type immunoelectrophoresis or any other immunological method well known to those skilled in the art.
Enfin, il est possible d'élaborer des anticorps contre les antigènes des lectines-facteurs d'attachement par des procédés connus, lesdits anticorps étant également utiles à titre de composants thérapeutiques et de réactifs pour le diagnostic.Finally, it is possible to develop antibodies against the antigens of the lectin-attachment factors by known methods, said antibodies also being useful as therapeutic components and reagents for diagnosis.
Les exemples suivants sont destinés à mettre en évidence d'autres caractéristiques et avantages de la présente invention.The following examples are intended to demonstrate other features and advantages of the present invention.
Sur les figures ci-annexées :
- - la figure 1 représente les immunoélectrophorèses quantitatives linaires de type "line-absorption" sur Meisseria gonorrhoeae pour les sérotypes :
- A figure 1a
- E figure 1b
- - la figure 2 représente des immunoélectrophorèses comme la figure 1 mais pour les sérotypes :
- C figure 2a
- D figure 2b
- - la figure 3 représente une chromatographie d'affinité d'un extrait protéique de Neisseria gonorrhoeae.
- FIG. 1 represents the linear line-absorption type quantitative immunoelectrophoresis on Meisseria gonorrhoeae for the serotypes:
- A figure 1a
- E figure 1b
- - Figure 2 shows immunoelectrophoresis as in Figure 1 but for serotypes:
- C figure 2a
- D figure 2b
- - Figure 3 shows an affinity chromatography of a protein extract of Neisseria gonorrhoeae.
Cet exemple a pour objet la mise en évidence des lectines-facteurs d'attachement décrits précédemment. Les sept sucres suivants ont été testés : maltose, galactosyl- amine,fucosylamine, glucosamine, acetylglucosamine, D galactosamine, thiogalactoside. Ces sucres ont été fixés sur des billes d'agarose par des techniques connues de l'homme de métier. Pour la mise en oeuvre de l'exemple proprement dit une suspensicn bactérienne de C,5 ml a été mise en contact avec C,5 ml de sucre immobilisé sur des billes. Après incubation, les billes ont été lavées puis colorées et observées au microscope. Suivant les sucres, deux cas se sont présentés. Pour les billes recouvertes d'a-D glucosamine ou de maltose, Neisseria gonorrhoeae adhérait sur ces billes. Par contre, pour les autres sucres, aucune adhésion du gonocoque n'était observée. Afin de mettre en évidence la spécificité de ces fixations, la possibilité d'inhiber la fixation du gonocoque a été étudiée en faisant incuber préalablement la suspension de Neisseria gonorrhoeae avec un sucre libre correspondant au sucre fixé mis en oeuvre dans l'étape suivante. Les résultats observés avec des souches de référence de deux collections et des souches sauvages sont identiques et mettent en évidence la fixation spécifique de ces lectines-facteurs d'attachement. La fixation était en effet inhibée uniquement si le sucre libre correspondant au mélange d'incubation étant ajouté en concentration suffisante (25 mg/ml). L'inhibition n'était réalisée avec aucun autre sucre. Donc, il existe à la surface du gonocoque des lectines-facteurs d'attachement qui permettent l'adhésion sur des récepteurs immobilisés. Ceux-ci possèdent entre autres des résidus glucosamine et maltose. Ceci permet la purification de ces lectines-facteurs par chromatographie d'affinité sur des récepteurs sucrés immobilisés.The purpose of this example is to demonstrate the lectins-attachment factors described above. The following seven sugars have been tested: maltose, galactosylamine, fucosylamine, glucosamine, acetylglucosamine, D galactosamine, thiogalactoside. These sugars were fixed on agarose beads by techniques known to those skilled in the art. For the implementation of the example proper, a bacterial suspension of C, 5 ml was brought into contact with C, 5 ml of sugar immobilized on beads. After incubation, the beads were washed then stained and observed under a microscope. According to the sugars, two cases arose. For beads coated with a-D glucosamine or maltose, Neisseria gonorrhoeae adhered to these beads. On the other hand, for the other sugars, no adhesion of the gonococcus was observed. In order to highlight the specificity of these fixations, the possibility of inhibiting the fixing of the gonococcus was studied by incubating beforehand the suspension of Neisseria gonorrhoeae with a free sugar corresponding to the fixed sugar used in the following step. The results observed with Reference strains from two collections and wild strains are identical and demonstrate the specific binding of these lectin-attachment factors. Fixation was in fact inhibited only if the free sugar corresponding to the incubation mixture was added in sufficient concentration (25 mg / ml). Inhibition was not achieved with any other sugar. Therefore, there are lectins-attachment factors on the surface of the gonococcus which allow adhesion to immobilized receptors. These have among others glucosamine and maltose residues. This allows the purification of these factor lectins by affinity chromatography on immobilized sugar receptors.
Les extraits antigéniques pariétaux solubilisés du gonocoque ont été soumis à une immunoélectrophorèse type "line-absorption". Les mélanges antigéniques étaient incorporés à des bandes d'agarose, ils migraient dans les gels contenant les antisérums polyvalents correspondants et quand un antigène rencontrait son anticorps spécifique à la con- centraticn appropriée du point d'équivalence, une ligne de précipitation était obtenue. Les figures 1 et 2 schématisent l'interprétation des résultats observés. Dans ces immunoélectrophorèses, les antigènes (Ag) étaient constitués par des extraits pariétaux (p) solubilisés de quatre souches de référence de gonocoque (GEIZER A, B, C, D), les anticorps (As) correspondaient aux antisérums spécifiques des extraits pariétaux. Les puits contenaient les sucres immobilisés suivants : puits 3 : glucosamine, puits 7 : galactosamine, puits 8 : maltose. Pour les quatre sérotypes de GEIZER testés, les résultats obtenus sont identiques : absorption des lignes de précipitation avec les sucres immobilisés maltose glucosarine uniquement.The extracts antigéni q ues parietal solubilized gonococcal were subjected to immuno-type "line-absorption." The antigenic mixtures were incorporated into agarose bands, they migrated into the gels containing the corresponding polyvalent antisera and when an antigen encountered its specific antibody at the appropriate concentration of the equivalence point, a precipitation line was obtained. Figures 1 and 2 schematically interpret the results observed. In these immunoelectrophoreses, the antigens (Ag) consisted of parietal extracts (p) solubilized from four reference gonococcal strains (GEIZER A, B, C, D), the antibodies (As) corresponded to the specific antisera of the parietal extracts. The wells contained the following immobilized sugars: well 3: glucosamine, well 7: galactosamine, well 8: maltose. For the four GEIZER serotypes tested, the results obtained are identical: absorption of the precipitation lines with immobilized sugars maltose glucosarin only.
Cet exemple montre la purification des facteurs d'attachement de Neisseria gonorrhoeae sérotype C de GEIZER réalisé par chromatographie d'affinité sur colonne agarose- glucosamine. Cette purification a été effectuée à partir de l'extrait protéique pariétal solubilisé du gonocoque : après fixation des lectines-facteurs d'attachement sur les récepteurs sucrés immobilisés et élimination dans le premier pic de la figure 3 des protéines n'interréagissant pas avec le sucre fixé, ces lectines-facteurs d'attachement étaient éluées par une solution de glucosamine à 0,25 M dans le deuxième pic. Ces lectines-facteurs d'attachement permettent de proposer :
- . des explications concernant les interactions hôte-gonocoque au niveau des cellules épithéliales de l'urètre et du rhinopharynx,
- . des thérapeutiques curatives mais surtout préventives en raison des propriétés antigéniques de ces facteurs d'attachement,
- . des techniques nouvelles de diagnostic des infections gonococciques.
- . explanations concerning host-gonococcal interactions in the epithelial cells of the urethra and the nasopharynx,
- . curative therapies but above all preventive because of the antigenic properties of these attachment factors,
- . new techniques for diagnosing gonococcal infections.
Claims (14)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8513066A FR2586563B1 (en) | 1985-09-03 | 1985-09-03 | NEISSERIA GONORRHOEAE ATTACHMENT LECTIN FACTORS, PROCESS FOR THEIR PREPARATION AND THERAPEUTIC COMPOSITION CONTAINING SUCH LECTINS OR CORRESPONDING ANTIBODIES OR ATTACHMENT ATTACHMENT RECEPTORS |
| FR8513066 | 1985-09-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0214057A1 true EP0214057A1 (en) | 1987-03-11 |
Family
ID=9322586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP86401916A Withdrawn EP0214057A1 (en) | 1985-09-03 | 1986-09-01 | Lectin attachment factors of Neisseria gonorrhoeae, method for their preparation and therapeutic composition containing these lectins, the corresponding antibodies or the corresponding attachment receptors |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0214057A1 (en) |
| JP (1) | JPS62187496A (en) |
| FR (1) | FR2586563B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5945514A (en) * | 1996-08-08 | 1999-08-31 | Mitsuru Akashi | Antiviral raw materials |
-
1985
- 1985-09-03 FR FR8513066A patent/FR2586563B1/en not_active Expired
-
1986
- 1986-09-01 EP EP86401916A patent/EP0214057A1/en not_active Withdrawn
- 1986-09-02 JP JP61206662A patent/JPS62187496A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| BIOLOGICAL ABSTRACTS, vol. 75, no. 2, 1983, résumé no. 11571, Philadelphia, P.A., US; E.R. GUBISH Jr. et al.: "Attachment of gonococcal pill to lectin-resistant clones of Chinese hamster ovary cells", & INFECT. IMMUN. 37(1): 189-194, 1982 * |
| CHEMICAL ABSTRACTS, vol. 103, no. 21, 25 novembre 1985, page 553, résumé no. 176837n, Columbus, Ohio, US; R.F. REST et al.: "Stimulation of human leukocytes by protein II+ gonococci is mediated by lectin-like gonococcal components", & INFECT. IMMUN. 1985, 50(1), 116-22 * |
| CHEMICAL ABSTRACTS, vol. 85, no. 3, 19 juillet 1976, page 449, résumé no. 18383s, Columbus, Ohio, US; A.N. JAMES et al.: "Attachment of gonococci to sperm. Influence of physical and chemical factors", & BR. J. VENER. DIS. 1976, 52(2), 128-35 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5945514A (en) * | 1996-08-08 | 1999-08-31 | Mitsuru Akashi | Antiviral raw materials |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62187496A (en) | 1987-08-15 |
| FR2586563B1 (en) | 1987-12-18 |
| FR2586563A1 (en) | 1987-03-06 |
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